Genetic labeling reveals temporal and spatial expression pattern of D2 dopamine receptor in rat forebrain

Abstract

The D2 dopamine receptor (Drd2) is implicated in several brain disorders such as schizophrenia, Parkinson's disease, and drug addiction. Drd2 is also the primary target of both antipsychotics and Parkinson's disease medications. Although the expression pattern of Drd2 is relatively well known in mouse brain, the temporal and spatial distribution of Drd2 is lesser clear in rat brain due to the lack of Drd2 reporter rat lines. Here, we used CRISPR/Cas9 techniques to generate two knockin rat lines: Drd2::Cre and Rosa26::loxp-stop-loxp-tdTomato. By crossing these two lines, we produced Drd2 reporter rats expressing the fluorescence protein tdTomato under the control of the endogenous Drd2 promoter. Using fluorescence imaging and unbiased stereology, we revealed the cellular expression pattern of Drd2 in adult and postnatal rat forebrain. Strikingly, the Drd2 expression pattern differs between Drd2 reporter rats and Drd2 reporter mice generated by BAC transgene in prefrontal cortex and hippocampus. These results provide fundamental information needed for the study of Drd2 function in rat forebrain. The Drd2::Cre rats generated here may represent a useful tool to study the function of neuronal populations expressing Drd2.

Keywords: Cerebral cortex; Drd2; Hippocampus; Knockin rats; Olfactory bulb.

Fig. 1

Generation of Drd2::Cre and Rosa26-LSL-tdTomato knockin rats and verification of Drd2 reporter rats. a Schematic diagram of the gene targeting strategy to insert the p2A-Cre cassette immediately before the stop codon of the Drd2 locus, between exons 7 and 3′ untranslational region (3′UTR). The p2A peptide will be cleaved and two independent protein DRD2 and CRE will be expressed. b Schematic diagram of the gene targeting strategy to insert the Cre reporter cassette into the Rosa26 locus between exon 1 and 2. The Cre reporter cassette is composed of CMV-IE enhancer/chicken β-actin/rabbit β-globin hybrid (CAG) promoter, a loxP (yellow triangles) flanked stop cassette, tdTomato red florescent protein, a woodchuck hepatitis virus post-translational regulatory element (WPRE), and a poly A tail. c Southern blot screen using EcoRV-digested genomic DNA and the 5′ probe indicated in the a. The wild-type and targeted Drd2 allele will yield a DNA fragment of 13.3 kb and 4.2 kb, respectively. d Southern blot screen using BglII-digested genomic DNA and the Cre probe indicated in the a. The targeted Drd2 allele will yield a DNA fragment of 9.1 kb. e Southern blot screen using Nde1-digested genomic DNA and the 5′ probe indicated in the d. The wild-type and targeted Rosa 26 allele will yield a DNA fragment of 5.6 kb and 3.5 kb, respectively. f Southern blot screen using BglII-digested genomic DNA and the WPRE probe indicated in the d. The targeted Rosa 26 allele will yield a DNA fragment of 5.2 kb. g Double fluorescence in situ hybridization (dFISH) of tdTomato and Drd2 mRNA in the striatum of Drd2 reporter rats. Scale bar, 100 µ m. h dFISH of tdTomato and Drd2 mRNA in the striatum of Drd1 reporter rats. Scale bar, 100 µm. i The enlarged image from the rectangle in g. Scale bar, 100 µm. j The enlarged image from the rectangle in h. Scale bar, 100 µm. k Immunofluorescent images of tdTomato and DRD2 in the striatum of Drd2 reporter rats. Arrows indicate the colocalization of tdTomato and DRD2. Scale bar, 25 µm. l Western blot of DRD2 and GAPDH in the striatum from Drd2::Cre^+/− and WT rats. Left, representative blots, right, quantification results. NS not significant, n = 3, t test

Fig. 2

Expression of tdTomato in the olfactory bulb (OB) of adult Drd2 reporter rats. a Diagram of rat brain sagittal section (top) and coronal section (bottom). The dashed line of the sagittal section diagram indicates the position of the coronal section. b Expression of tdTomato in OB. Scale bar, 400 µm. c Immunofluorescent image of tdTomato, OMP, and DAPI in the olfactory epithelial layer. Arrows indicate Drd2⁺ olfactory sensory neurons. Scale bar, 20 µm. d The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the OB. e Expression of tdTomato when injection of AAV-LSL-tdTomato into the GCL of adult Drd2::Cre rats. Scale bar, 500 µm. f Expression of tdTomato in GL. Scale bar, 200 µm. g Immunofluorescent image of tdTomato, TH, and GAD67 from the rectangle in f. Scale bar, 40 µm. Arrows indicate Drd2⁺ cells expressing both TH and GAD67, arrowheads indicate Drd2⁺ cells only expressing TH and empty arrows indicate Drd2⁺ cells expressing neither TH nor GAD67. h Immunofluorescent image of tdTomato and TBX 21 in the MCL. Scale bar, 25 µm. i Immunofluorescent image of tdTomato and GAD67 in the GCL. Scale bar, 25 µm. ONL olfactory nerve layer, GL glomerular layer, EPL external plexiform layer, MCL mitral cell layer, GCL granular cell layer, RMS rostral migratory stream

Fig. 3

Expression of tdTomato in the prefrontal cortex (PFC) of Drd2 reporter rats. a Diagram of rat brain sagittal section (top) and coronal section (bottom). The dashed line of the sagittal section diagram indicates the position of the coronal section. b Expression of tdTomato in the PFC. Scale bar, 500 µm. c, d Enlarged images from the rectangles in b. Scale bar, 200 µm for c, 100 µm for d. e The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the M2. f The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the PrL. g Expression of tdTomato and GAD67 in M2. Scale bar, 100 µm. h, i Immunofluorescent images of tdTomato, GAD67, NeuN, and DAPI from the rectangles in panel G. Arrows indicate Drd2⁺ GABAergic interneurons. Scale bar, 40 µm. j The percentage of Drd2⁺ neurons among all neurons in the M2. ***p < 0.001, n = 3, t test. k The percentage of GABAergic interneurons in the Drd2⁺ neurons in the M2. ***p < 0.001, n = 3, t test. l Expression of tdTomato and PV in M2. Scale bar, 100 µm. m, n, Immunofluorescent images of tdTomato, PV, NeuN, and DAPI from the rectangles in panel L. Arrows indicate Drd2⁺ PV-positive interneurons. Scale bar, 40 µm. M2 secondary motor cortex, PrL prelimbic cortex, MO medial orbital cortex, VO ventral orbital cortex, LO lateral orbital cortex, DLO dorsolateral orbital cortex

Fig. 4

Expression of tdTomato in the SS1 and RS of Drd2 reporter rats. a Diagram of rat brain sagittal section (top) and coronal section (bottom). The dashed line of the sagittal section diagram indicates the position of the coronal section. The red and blue rectangles indicate the brain regions shown in b, g. b Immunofluorescent images of tdTomato, GAD67 and NeuN in SS1. Scale bar, 100 µm. c The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the SS1. d, e Immunofluorescent images of tdTomato, GAD67, and NeuN from the rectangles in b. Arrows indicate Drd2⁺ GABAergic interneurons. Scale bar, 50 µm. f Enlarged image from the rectangle in b. Scale bar, 40 µm. g Expression of tdTomato in RS. Scale bar, 200 µm. h Immunofluorescent images of tdTomato, GAD67, and NeuN from the rectangle in g. Arrows indicate Drd2⁺ GABAergic interneurons. Scale bar, 50 µm. SS1: primary somatosensory cortex, RS retrosplenial cortex

Fig. 5

Expression of tdTomato in the ACC of Drd2 reporter rats. a Diagram of rat brain sagittal section (top) and coronal section (bottom). The dashed line of the sagittal section diagram indicates the position of the coronal section. The rectangle indicates the brain regions shown in b. b Expression of tdTomato. Scale bar, 500 µm. c Expression of tdTomato from the rectangle in b. Scale bar, 200 µm. d, e Immunofluorescent images of tdTomato, GAD67, and NeuN from the rectangles in c. Arrows indicate Drd2⁺ GABAergic interneurons. Scale bar, 40 µm. ACC anterior cingulate cortex

Fig. 6

Expression of tdTomato in the hippocampus of Drd2 reporter rats. a Immunofluorescent images of tdTomato and GAD67 in the dorsal hippocampus. Scale bar, 200 µm. b Enlarged images of the rectangle in a. Arrows indicate Drd2⁺ GABAergic interneurons. Empty arrows indicate Drd2⁺ cells negative for GAD67. Scale bar, 100 µm. c The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the dorsal hippocampus. Expression of tdTomato in the slm of CA1 region of dorsal (d) and ventral (e) hippocampus after injecting AAV-LSL-tdTomato into the entorhinal cortex. Scale bar, 1000 µm. f Expression of tdTomato in the ventral hippocampus. Scale bar, 1000 µm. g, h Enlarged images of the rectangles in f. Scale bar, 100 µm. i Immunofluorescent images of DRD2 and GAD67 in the ventral hippocampus. Arrows indicate Drd2⁺ GABAergic interneurons. Scale bar, 50 µm. so stratum oriens, sp stratum pyramidale, sr stratum radiatum, slm stratum lacunosum, osm outer stratum moleculare, ism inter stratum moleculare, gr granule cell layer, h hilus

Fig. 7

The Drd2⁺ cells in the olfactory bulb (OB), prefrontal cortex (PFC), second visual cortex (V2), entorhinal cortex (EC), and hippocampus of different aged Drd2 reporter rats. a Expression of tdTomato in the OB. Scale bar, 500 µm. b The percentage of Drd2⁺ granule cells among total granule cells in the OB. NS not significant, **p < 0.01, *p < 0.05, n = 3, one-way ANOVA. c Expression of tdTomato in the PFC. Scale bar, 500 µm. d The percentage of Drd2⁺ neurons among total neurons in the layer 2–3 and 5–6 of PFC. ***p < 0.001, n = 3, two-way ANOVA. e Expression of tdTomato in the V2, EC and hippocampus. Scale bar, 1000 µm. f The percentage of Drd2⁺ neurons among total neurons in the layer 2–3 and 5–6 of V2. **p < 0.01, n = 3, two-way ANOVA. g the number of Drd2⁺ neurons in the ventral hippocampus as indicated in e. NS not significant, ***p < 0.001, n = 3, two-way ANOVA

Fig. 8

Expression of tdTomato in the PFC and hippocampus of adult Drd2 reporter mice. a Diagram of mouse brain sagittal section (top) and coronal section (bottom). The dashed line of the sagittal section diagram indicates the position of the coronal section. b Expression of tdTomato in the PFC of Drd2 reporter mice. Scale bar, 500 µm. c, d Enlarged images from the rectangles in b. Scale bar, 200 µm for c, 100 µm for d. e The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the M2. f The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the PrL. g Immunofluorescent images of tdTomato and GAD67::GFP in the dorsal hippocampus. The Drd2 reporter mice were crossed with GAD67::GFP mice to obtain this figure. Scale bar, 200 µm. h The percentage of Drd2⁺ cells in different layers among total Drd2⁺ cells in the dorsal hippocampus. i Enlarged images of the rectangle in g. Arrows indicate Drd2⁺ GABAergic interneurons. Empty arrows indicate Drd2⁺ cells negative for GAD67. Scale bar, 100 µm. j Expression of tdTomato and GAD67::GFP in the ventral hippocampus. Scale bar, 1000 µm. k Enlarged image from the rectangle in j. Scale bar, 100 µm